Publications referred to by reference numbering in this specification correspond to the reference list at the end of the specification are hereby incorporated by reference in their entirety.
Various ways of measuring rates of synthesis or breakdown of biological polymers and other biological molecules of interest have been described. One such invasive method involves the collection of tissues by various invasive procedures, e.g., surgical excision, percutaneous biopsy, post-mortem analysis, or other sampling procedures (termed “invasive” procedures herein), after administration of an isotopically labeled precursor molecule, then isolation of the polymer or other molecule of interest from the tissue so collected, followed by measurement of the isotopic content or labeling pattern in said polymer or other molecule and calculation of the synthesis or breakdown rate of said polymer or other molecule based on the rate of isotope incorporation. This method has some disadvantages that include, inter alia, the need for invasive tissue measurements with attendant medical risk, discomfort, need for expert medical involvement, and limitations on the number of measurements that can be performed.
Another invasive method involves repeated collection of tissue by the invasive procedures listed above after an intervention with measurement of the content (concentration or pool size) of the polymer or other molecule of interest in each tissue sample, and calculation of the rate of change in the net pool size over time, thereby determining the net synthesis (accrual) or net breakdown (depletion) rate. This method has some disadvantages that include, inter alia, the need for repeated measurements and the lack of a true synthetic or breakdown rate measured, with instead a net accrual or depletion rate generated. Further, it is a well-recognized principle in biochemistry that net changes in concentration (accrual or depletion) are not identical to and do not reveal true or absolute rates of synthesis or breakdown (7), because concurrent synthesis and breakdown (herein termed “turnover”) is not measured or accounted for by net changes in concentration.
A method that is commonly used in medical practice involves the indirect estimate of pool size or concentration, and their changes over time, by use of repeated radiographic measurements (e.g., x-rays or dual-energy-X-ray absorptiometry for estimating bone mass (4); nuclear magnetic resonance imaging or computerized tomography for estimating muscle or fat mass (9); radiographic procedures for estimating tumor mass). This approach suffers from the same limitations as direct biochemical measurements of concentrations or pool sizes of molecules (noted above), in addition to limitations of accuracy.
Another non-invasive method that has been used involves the collection of a breakdown product that is specific for and derived from a biological molecule or other molecule of interest and that is secreted or excreted into blood or urine, and calculation of the breakdown rate of the biological molecule or other molecule based on the recovery of said breakdown product. (10, 11). This method has some disadvantages that include, inter alia, the inability to measure synthesis rates or true breakdown rates, rather than a net release rate, and other technical limitations that are well-described for these methods (e.g., incomplete recovery of breakdown products due to their biological clearance and catabolism in the organism; interference by delayed or unpredictable excretion of the breakdown products; etc).
The disadvantages and limitations of these prior methods for measuring the synthesis and breakdown rates of biological molecules located in inaccessible tissues inaccessible biological samples are substantial and have held back important fields, including medical diagnostics, drug discovery, genetics, functional genomics and basic research. The disadvantages noted here are not intended to be comprehensive; many other limitations and disadvantages of these methods exist and could be mentioned.
An optimal non-invasive method of measuring rates of biosynthesis and breakdown rates of biological molecules would have the following characteristics: accuracy, capacity to measure true or absolute rates of biosynthesis or breakdown (i.e., accounts for turnover), and does not require total quantitative collection of breakdown products (i.e. metabolic derivatives and catabolic products). Furthermore, an ideal method would allow constant isotope levels in the precursor pool to be maintained for prolonged periods of time in a simple, non-demanding manner, for example, on the order of a few half-lives of long-lived molecules. However, there has not been a technique that has fulfilled these objectives. A method for measuring non-invasively the rates of synthesis or breakdown of biological molecules that are inaccessible or not easily accessible to direct sampling (e.g. molecules in or associated with tissues of the internal organs) and that is widely applicable, reliable, easy to perform, inexpensive, without toxicities or complications, applicable in human subjects, free of the need for medical supervision or in-patient procedures (such as intravenous infusions), does not require complex instructions, and possesses the advantages of simple interpretation, therefore would be extremely valuable and useful in fields ranging from medical diagnostics to drug discovery, genetics, functional genomics, and basic research.